Unit 3 Exam lecture 18 Flashcards
Restriction enzymes do what
recognize specific sequences and cut the DNA at defined sites
restriction enzymes leave what two types of ends
blunt or sticky ends
HindII leaves what kind of ends
sticky
PvuII leaves what kind of ends
blunt
How do we find and isolate a gene of interest
using restriction enzymes
How to we separate the gene of interest that we want to study
use of souther blotting or restriction enzymes to isolate
What is the overall process of southern blotting
- digest genome with restriction enzyme
- separate on gel
- use a complementary probe to isolate the gene of interest
- autoradiography to detect fragments on probes
Southern blotting detects what
Northern blotting detects what
Western blotting detects what
- DNA
- RNA
- Protein
what is the caveat of southern blotting
need to have probe that is complementary otherwise will not be able to isolate
What are the three things that you need in a cloning vector to insert a genome
- origin of replication recognized in host cell
- selectable markers
- single cleavage site for each restrictive enzyme used
What is the process of inserting foreign DNA into the plasmid
- plasmid and foreign DNA are cut by same restriction enzyme
- when mixed sticky ends anneal
- Nicks sealed by DNA ligase
How is the plasmid with foreign DNA placed into the cell
transformation
what are the effects of inserting foreign DNA into the middle of the lacZ gene
when grown on special gel, bacteria with recombinant plasmid will not synthesize B-galactosidase and their colonies will be white while the other will be blue
benefit of including a marker with antibiotic resistance
only cells which take up the plasmid will be able to survive and proliferate
What are the steps in PCR
- Heating to separate
- Annealing of primers
- Elongation
repeat
What are the limitations of PCR
need primers that are specific to the sequence you want to proliferate, primers could anneal to the wrong DNA
What was the original way that DNA was sequenced
Used ddNTPs which lack 3’ OH group,
four reactions each with a different base ddNTP,
all have the 4 deoxyribonucleotide triphosphates,
when ddNTP incorperated into genome chain stops,
get different lengthed fragments,
run 4 reaction results through a gel to determine complementary sequence to the original strand.
Describe the innovation and design of the Sanger Sequencing method
single stranded DNA fragment
four ddNTPs tagged with different flourescent dyes
primer added and chain extended
products denatured and fragments run in gel
DNA is detected by laser beam to determine order of sequence
computer takes data and converts it to target sequence
What is the process of Illumina sequencing
Place entire genome
restriction enzymes cut it up
add specific addaptors for one primer to fragments
single primer type added
primer binds to adaptor
only need one primer and adaptor for everything
What is a contig
alignment of DNA fragments at the points where they overlap to make consensus contig (real sequence of genome)
Forward genetics are what
starting with phenotype and proceeding to genotype
Reverse genetics are what
starting with genotype and altering the sequence to determine effects on phenotype
What is site directed mutagenesis
creation of transgenic animals by adding sequence of interest to genome of organisms that normally lacks sequence
what is benefit to using mammals for site directed mutagenesis
oocytes are large enough that DNA can be injected directly
Explain the process of creating transgenic mice
Euthinized males have sex with females to get hormones going but no sperm
foreign DNA injected into pronuclei
embryos implanted in pseudopregnant female
offspring are tested for presence of transgene
What is the success rate of creating transgenic organisms
low, 10-30% of eggs survive and most do not have copy
What is the point of inserting foreign DNA early in development when creating a transgenic animal
so most if not all copies of chromosomes have the foreign DNA
What does the target sequence of DNA need for CRISPR-Cas9 to work
complementation with the sgRNA and need for PAM
What happens to the DNA after it is cleaved by Cas9
fragments will either join together or their will be new donor DNA inserted
Why would it be better to change Cas9 to make single stranded breaks
easier to insert new DNA than double stranded breaks
When should genes be inserted or destroyed while using Cas9
Early in development or end up with mosaics
What are the limitations to CRISPR-Cas9
Off target cleavage, sgRNA doesnt have to match 100%
siRNAs do what
switch off gene expression
siRNAs need to be recognized and cleaved by
Dicer
Why can siRNAs not be used on gene families
homology is too similar and siRNA will not be specific enough
ApoB is a key part of what
lipoproteins
genetic mutations that cause elevated levels of ApoB cause
increased chance of coronary artery disease
How to potentially combat elevated levels of ApoB
RNAi has been demonstrated to be able to reduce levels of ApoB
What is one reason that using synthesized siRNAs can be difficult (evident in monkey study)
need to find way to get them into cell
How were scientists able to get ApoB-siRNAs into primate cells
encapsulated ApoB-siRNAs in lipids
